BK beta subunits of Slo family potassium channels

ABSTRACT

The invention provides isolated nucleic acid and amino sequences of BK beta 2, BK beta 3, and BK beta 4, antibodies to the BK beta subunits, methods of detecting the BK beta subunits, methods of screening for modulators of Slo family potassium channels comprising BK beta subunits.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase under 35 U.S.C. §371 ofPCT/US00/04441, which was filed Feb. 22, 2000, and claims priority toU.S. Ser. No. 60/121,224, filed Feb. 23, 1999, and U.S. Ser. No.60/163,367, filed Nov. 3, 1999, herein both incorporated by reference intheir entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofBK beta subunits 2, 3, and 4, which are auxiliary subunits of Slopotassium channels, antibodies to BK beta subunits 2–4, methods ofdetecting BK beta subunits 24, methods of screening for modulators ofSlo potassium channels comprising BK beta subunits 2, 3, or 4, and kitsfor screening for modulators of Slo family potassium channels comprisingBK beta subunits 2, 3, or 4.

BACKGROUND OF THE INVENTION

Potassium channels are involved in a number of physiological processes,including regulation of heartbeat, dilation of arteries, release ofinsulin, excitability of nerve cells, and regulation of renalelectrolyte transport. Potassium channels are thus found in a widevariety of animal cells such as nervous, muscular, glandular, immune,reproductive, and epithelial tissue. These channels allow the flow ofpotassium in and/or out of the cell under certain conditions. Forexample, the outward flow of potassium ions upon opening of thesechannels makes the interior of the cell more negative, counteractingdepolarizing voltages applied to the cell. These channels are regulated,e.g., by calcium sensitivity, voltage-gating, second messengers,extracellular ligands, and ATP-sensitivity.

Potassium channels are made by alpha subunits that fall into 8 families,based on predicted structural and functional similarities (Wei et al.,Neuropharmacology 35(7):805–829 (1997)). Three of these families (Kv,eag-related, and KQT) share a common motif of six transmembrane domainsand are primarily gated by voltage. Two other families, CNG and SK/IK,also contain this motif but are gated by cyclic nucleotides and calcium,respectively. The three other families of potassium channel alphasubunits have distinct patterns of transmembrane domains. Slo familypotassium channels, or BK channels have seven transmembrane domains(Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94(25):14066–71 (1997)) andare gated by both voltage and calcium or pH (Schreiber et al., J. Biol.Chem. 273:3509–16 (1998)). Another family, the inward rectifierpotassium channels (Kir), belong to a structural family containing 2transmembrane domains, and an eighth functionally diverse family (TP, or“two-pore”) contains 2 tandem repeats of this inward rectifier motif.

Potassium channels are typically formed by four alpha subunits, and canbe homomeric (made of identical alpha subunits) or heteromeric (made oftwo or more distinct types of alpha subunits). In addition, potassiumchannels made from Kv, KQT and Slo or BK subunits have often been foundto contain additional, structurally distinct auxiliary, or beta,subunits. These subunits do not form potassium channels themselves, butinstead they act as auxiliary subunits to modify the functionalproperties of channels formed by alpha subunits. For example, the Kvbeta subunits are cytoplasmic and are known to increase the surfaceexpression of Kv channels and/or modify inactivation kinetics of thechannel (Heinemann et al., J. Physiol. 493:625–633 (1996); Shi et al.,Neuron 16(4):843–852 (1996)). In another example, the KQT family betasubunit, minK, primarily changes activation kinetics (Sanguinetti etal., Nature 384:80–83 (1996)).

Slo or BK potassium channels are large conductance potassium channelsfound in a wide variety of tissues, both in the central nervous systemand periphery. They play a key role in the regulation of processes suchas neuronal integration, muscular contraction and hormone secretion.They may also be involved in processes such as lymphocytedifferentiation and cell proliferation, spermatocyte differentiation andsperm motility. Three alpha subunits of the Slo family have been cloned,i.e., Slo1, Slo2, and Slo3 (Butler et al., Science 261:221–224 (1993);Schreiber et al., J. Biol. Chem., 273:3509–16 (1998); and Joiner et al.,Nature Neurosci. 1:462–469 (1998)). These Slo family members have beenshown to be voltage and/or calcium gated, and/or regulated byintracellular pH.

A BK (or Slo) beta subunit that associates with Slo/BK potassiumchannels has been cloned and called BK beta 1 (Dworetzky et al., J.Neurosci. 16:4543–50 (1996); U.S. Pat. No. 5,776,734; see also Xia etal., J. Neurosci. 19:5255–5264 (1999), Wallner et al., Proc. Nat'l Acad.Sci. USA 96:4137–4142 (1999), Ali Riazi et al., Genomics 62:90–94(1999), and EP 0 936 271 A1). BK beta 1 has short cytoplasmic N and Ctermini and has two membrane-spanning regions with a large extracellularloop. Functionally, BK beta 1 modulates the activation kinetics andcalcium sensitivity of Slo1 channels (McManus et al., Neuron 14:645–50(1995)). BK beta 1 also increases calcium sensitivity, sensitivity toextracellular toxins, and decreases the activation rate for Slo1channels.

Additional beta subunits for Slo family potassium channels remain to beidentified. The discovery and characterization of those Slo or BK betasubunits will provide important insights into how Slo potassium channelsfunction in different environments, and how they respond to variousactivation mechanisms. Such information would also allow theidentification of modulators of Slo potassium channels and the use ofsuch modulators as therapeutic agents.

SUMMARY OF THE INVENTION

The present invention thus identifies for the first time BK beta 2, BKbeta 3, and BK beta 4, each of which is a beta subunit of a Slo familypotassium channel. These BK beta subunits have neither been previouslycloned nor identified, and the present invention provides both the aminoacid and nucleotide sequences of BK beta 2, BK beta 3, and BK beta 4.

In one aspect, the present invention provides an isolated nucleic acidencoding a beta subunit of a potassium channel, wherein the betasubunit: (i) forms, with at least one alpha unit, a Slo potassiumchannel; (ii) comprises an amino acid sequence that has greater thanabout 70% identity to the S1–S2 region of BK beta 2, BK beta 3, or BKbeta 4; and (iii) specifically binds to polyclonal antibodies generatedagainst SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO:5.

In one aspect, the present invention provides an isolated nucleic acidencoding a beta subunit of a potassium channel, wherein the betasubunit: (i) forms, with at least one alpha subunit, a Slo potassiumchannel; (ii) comprises an amino acid sequence that has greater thanabout 70% identity to the S1–S2 region of BK beta 2, BK beta 3, or BKbeta 4; and (iii) specifically binds to polyclonal antibodies generatedagainst SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; wherein said nucleicacid either: (i) selectively hybridizes under moderately stringenthybridization conditions to a nucleotide sequence of SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:6; or (ii) encodes a protein which could be encodedby a nucleic acid that selectively hybridizes under moderately stringenthybridization conditions to a nucleotide of SEQ ID NO:2, SEQ ID NO:4, orSEQ ID NO:6.

In one embodiment, the nucleic acid is amplified by primers thatselectively hybridize under stringent hybridization conditions to thesame sequence as primers selected from the group consisting of:

5-ATGACAGCCTTTCCTGCCTCAGGGAAG-3 (SEQ ID NO:7)5-AGATTTCTCTGCTCTTCCTTTGCTCCTCC-3 (SEQ ID NO:8)5-GGCTGGCTGGACTGTAGAAGCATG-3 (SEQ ID NO:9)5-GAGGCTGTCCAGATAAATCCCAAGTGC-3 (SEQ ID NO:10)5-GGACTGAGAAGCCCATCATGGCAAACC-3 (SEQ ID NO:11);5-ATGGCGAAGCTCCGGGTGGCTTAC-3 (SEQ ID NO:12)5-TTAAGAGAACTTGCGCTTCTTCATGG-3 (SEQ ID NO:13)5-GATGTGCTTCTGCATCGCACTCATG-3 (SEQ ID NO:14); and5-AAGATGTCGATATGGACCAGTGGCC-3 (SEQ ID NO:15)5-TTATCTATTGATCCGTTGGATCCTCTC-3 (SEQ ID NO:16)5-CTCCTTCAGCTGTCCTCCAGACTGC-3 (SEQ ID NO:17)5-GTCCCAGTAGAATAGCTCGGTCCTC-3 (SEQ ID NO:18).

In one embodiment, the nucleic acid encodes a beta subunit having amolecular weight of about between 24–34 kDa, 18–28 kDa, or 22–32 kDa. Inanother embodiment, the nucleic acid encodes human BK beta 2, 3, or 4.In another embodiment, the nucleic acid encodes SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:5. In another embodiment, the nucleic acid has anucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

In another aspect, the present invention provides an isolated betasubunit of a potassium channel, wherein the beta subunit: (i) forms,with at least one alpha subunit, a Slo potassium channel; (ii) comprisesan amino acid sequence that has greater than about 70% identity to theS1–S2 region of BK beta 2, BK beta 3, or BK beta 4; and (iii)specifically binds to polyclonal antibodies generated against SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5.

In one embodiment, the beta subunit is human BK beta 2, 3, or 4. Inanother embodiment, the beta subunit has the sequence of SEQ ID NO:1,SEQ ID NO:3, or SEQ ID NO:5.

In another aspect, the present invention provides an antibody thatselectively binds to a beta subunit as described above.

In another aspect, the present invention provides an expression vectorcomprising a nucleic acid as described above.

In another aspect, the present invention provides host cell transfectedwith an expression vector as described above.

In another aspect, the present invention provides a method foridentifying a compound that increases or decreases ion flux through apotassium channel, the method comprising the steps of: (i) contactingthe compound with a eukaryotic host cell or cell membrane in which hasbeen expressed potassium channel comprising a beta subunit, wherein thebeta subunit: (a) forms, with at least one alpha subunit, a Slopotassium channel; (b) comprises an amino acid sequence that has greaterthan about 70% identity to the S1–S2 region of a BK beta 2, BK beta 3,or a BK beta 4; and (c) specifically binds to polyclonal antibodiesgenerated against SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; and (ii)determining the functional effect of the compound upon the cell or cellmembrane expressing the Slo potassium channel.

In one embodiment, the functional effect is determined by measuringchanges in current or voltage. In another embodiment, the beta subunitis recombinant. In one embodiment, the beta subunit is human BK beta 2,3, or 4. In another embodiment, the beta subunit has the sequence of SEQID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

In another aspect, the present invention provides a method of detectingthe presence of BK beta 2, 3 or 4 in a sample, the method comprising thesteps of: (i) isolating a biological sample; (ii) contacting thebiological sample with a BK beta 2, 3, or 4-specific reagent thatselectively associates with BK beta 2, 3, or 4; and, (iii) detecting thelevel of BK beta 2, 3, or 4-specific reagent that selectively associateswith the sample.

In one embodiment, the BK beta specific reagent is selected from thegroup consisting of: BK beta specific antibodies, BK beta specificoligonucleotide primers, and BK beta specific-nucleic acid probes. Inanother embodiment, the sample is from a human.

In another aspect, the present invention provides, in a computer system,a method of screening for mutations of human BK beta 2, BK beta 3, or BKbeta 4 genes, the method comprising the steps of: (i) entering into thesystem at least about 25 nucleotides of first nucleic acid sequenceencoding a beta subunit having a nucleotide sequence of SEQ ID NO:2, SEQID NO:4, or SEQ ID NO:6; (ii) comparing the first nucleic acid sequencewith a second nucleic acid sequence having substantial identity to thefirst nucleic acid sequence; and (iii) identifying nucleotidedifferences between the first and second nucleic acid sequences.

In one embodiment, the second nucleic acid sequence is associated with adisease state.

In aspect, the present invention provides a computer readable substratecomprising the first nucleic acid sequence.

In another aspect, the present invention provides, in a computer system,a method for identifying a three-dimensional structure of BK beta 2, BKbeta 3, or BK beta 4 subunits, the method comprising the steps of: (i)entering into the system an amino acid sequence of at least 25 aminoacids of a beta subunit or at least 75 nucleotides of a gene encodingthe beta subunit, the beta subunit having an amino acid sequence of SEQID NO:1, SEQ ID NO:3, or SEQ ID NO:5; and (ii) generating athree-dimensional structure of the beta subunit encoded by the aminoacid sequence.

In another aspect, the present invention provides a computer readablesubstrate comprising the three dimensional structure of the betasubunit.

In one embodiment, the amino acid sequence is a primary structure andwherein said generating step includes the steps of: (i) forming asecondary structure from said primary structure using energy termsdetermined by the primary structure; and (ii) forming a tertiarystructure from said secondary structure using energy terms determined bysaid secondary structure. In another embodiment, said generating stepfurther includes the step of forming a quaternary structure from saidtertiary structure using anisotropic terms encoded by the tertiarystructure. In another embodiment, the method further comprises the stepof identifying regions of the three-dimensional structure of a BK beta2, BK beta 3 or BK beta 4 subunit that bind to ligands and using theregions to identify ligands that bind to the beta subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Alignment of the deduced amino acid sequence of human BK beta 1,2, 3, and 4. Residues conserved in three or more proteins are shaded.Gaps in the alignment are indicated by dashes. Amino acid numbers aregiven at the left margin. Two predicted transmembrane domains areoutlined in black. The Genbank accession number for the BK beta 1sequence is U38907 (Dworetsky et al., J. Neurosci. 16:4543–4550 (1996).

FIG. 2: Messenger RNA expression patterns for human BK beta 2, 3, and 4.Height of bars indicates expression level relative to the highestexpression level seen for that gene. All data comes from hybridizationof ³²P-labeled cDNA probes against human mRNA masterblots (Clontech).

FIG. 3A: Normalized conductance-voltage curves for hSlo1 in the absence(dashed lines) or the presence (solid lines) of BK beta 3. Data is frominside-out membrane patches of Xenopus oocytes expressing Slo1.Conductance-voltage curves were derived from the current voltagerelationship according to: G=I/V−Ek, where Ek was omV. Current voltagerelationships were determined with slow voltage ramps (1.5 s) from −150mV to +100 mV. Free calcium concentration was calculated to be 7 μM.

FIG. 3B: V1/2 activation values were calculated from G-V as shown inFIG. 3A. Mean V1/2act +/−sem values are plotted against calculated freecalcium concentration for hSlo1 in the absence (squares) and presence(diamonds) of BK beta 3. Symbols are means of 3–4 patches per point.

FIG. 4: Effect of BK beta 3 on voltage- and calcium dependent activationof Slo-1. Conductance-voltage relationships were constructed bymeasuring the deactivating tail current amplitude at −50 mV following aseries of 500 ms depolarizing steps (−100 mV to +150 mV in 10 mVincrements) from a holding potential of −140 mV. Activation curves weregenerated by plotting normalized tail current amplitude against the steppotential, fitted with a Boltzman distribution and are plotted in FIGS.4A–4C for Slo-1 alone (solid circles) and Slo-1 co-expressed with BKbeta 3 (open squares). The voltages for half-maximal activation(V1/2act, mV) were determined from fitted data and are plotted againstfree calcium concentration in FIG. 4D. BK beta 3 had no significanteffect on the voltage-dependence of Slo-1 activation in the presence oflow Ca2+ concentrations (10 μM or less, FIGS. 4A, B and D). In thepresence of 50 μM free Ca2+ however, BK beta 3 produced a small, butsignificant (approximately 15 mV), leftward shift in the Slo-1activation curve (FIGS. 4C, D). Symbols represent the mean of 8–12experiments, vertical lines represent the S.E.M. * indicates P<0.05,2-tail t-test.

FIG. 5. Effect of BK beta 3 on the time-course of Slo-1 activation.Voltage clamp records show Slo-1 current activation, in the absence (A)or presence (B) of BK beta 3, following a series of 500 ms depolarizingsteps (−100 mV to +150 mV in 10 mV increments) from a holding potentialof −140 mV. Free Ca2+ was 10 μM, scale bars represent 1 nA and 10 ms.Currents elicited by depolarizing voltage steps were fit with a singleexponential function. Activation rates (τ act, ms) were determined inintracellular solutions containing either 1 μM (squares) or 10 μM(triangles) free Ca2+, in the absence (solid symbols) or presence (opensymbols) of BK beta 3. BK beta 3 produced a slowing of activation at allvoltages in the presence of 1 μM and 10 μM free Ca2+. Symbols representthe mean of 8–12 experiments, vertical lines represent the S.E.M.

FIG. 6. Slo-1/BKB3 forms BK type II-like charybdotoxin-(CTX) insensitivechannels. Slo-1 (A) or Slo-1+BK beta 3 (B) were expressed in HEK293cells and membrane currents measured using the whole-cell voltage-clamptechnique. Currents were elicited with 250 ms depolarizing steps (to −20mV in the examples shown) from a holding potential of −80 mV. Asillustrated in FIG. 6A, membrane currents in cells expressing Slo-1alone were strongly inhibited by both TEA (5 mM) and CTX (100 nM). Incontrast, membrane currents in cells co-expressing Slo-1 and BK beta 3were TEA (5 mM) sensitive but CTX (100 nM) insensitive (FIG. 6B).Similar recordings were made from 4 cells expressing Slo-1 alone and 6cells co-expressing Slo-1/BKB3. Scale bars represent 1 nA and 200 ms.

FIG. 7. Whole-cell recording from a CHO cell cotransfected with BKB4 andSlo1. Cell was stepped to the indicated voltages for 400 ms followed bya step to −20 mV for 40 ms, from a holding potential was −80 mV. Unlikehomomeric Slo1 channels, the BKB4+Slo1 channels inactivate rapidly.Pipette solution contained (mM): 40 KCl, 100 KF, 5 NaCl, 1 MgCl2, 5glucose, 10 HEPES, pH=7.4. Extracellular bath solution contained (mM):138 NaCl, 2 CaCl2, 5.4 KCl, 1 MgCl2, 10 glucose, 10 HEPES, pH=7.4.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides for the first time nucleic acids encodingnovel potassium channel beta subunits BK beta 2, BK beta 3, and BK beta4, identified and cloned from human tissues. These beta subunits arepolypeptide monomers having two transmembrane domains separated by alarge extracellular linker. BK beta subunits associate with alphasubunit monomers from the Slo gene family to produce Slo potassiumchannels. The larger Slo family alpha polypeptide monomers contain sixor seven transmembrane domains that form the ion-conducting core of aSlo potassium channel, while the BK beta monomers regulate channelkinetics, expression levels, and sensitivity to bound ligands. BecauseSlo potassium channels can open at subthreshold voltages in the presenceof sufficient intracellular calcium, they have significant roles inmaintaining the resting potential and in controlling excitability of acell. Altering the calcium sensitivity, kinetics, and expression levelsof these Slo channels with BK beta subunits is likely to result inchanges in firing frequency and secretion levels.

The invention also provides methods of screening for modulators of Slopotassium channels comprising a BK beta subunit 2, 3, or 4. Because ofthe importance of Slo channels to firing frequency and secretion, suchmodulators of Slo channel activity are useful for treating CNS disorderssuch as migraines, hearing and vision problems, learning and memoryproblems, learning and memory problems, seizures, psychotic disorders,and as neuroprotective agents (e.g., to prevent stroke). They are alsouseful in treating disorders of vascular and muscle tone, breathing(asthma), hormone secretion, spermatocyte differentiation and motility,lymphocyte differentiation and cell proliferation.

Furthermore, the invention provides assays for a Slo potassium channelactivity, where the channel comprising BK beta subunit 2, 3, or 4 actsas a direct or indirect reporter molecule. Such uses of the Slopotassium channel as a reporter molecule in assay and detection systemshave broad applications, e.g., the Slo potassium channel can be used asa reporter molecule to measure changes in potassium concentration,membrane potential, current flow, ion flux, transcription, signaltransduction, receptor-ligand interactions, second messengerconcentrations, in vitro, in vivo, and ex vivo. In one embodiment, theSlo potassium channel can be used as an indicator of current flow in aparticular direction (e.g., outward or inward potassium flow), and inanother embodiment, the Slo potassium channel can be used as an indirectreporter via attachment to a second reporter molecule such as greenfluorescent protein.

Finally, the invention provides for methods of detecting BK betasubunits 2–4 nucleic acid and protein expression, allowing investigationof the channel diversity provided by the beta subunits and theregulation/modulation of channel activity provided by the beta subunits,as well as diagnosis of disease involving abnormal ion flux, includingdiagnosis of CNS disorders such as migraines, hearing and visionproblems, learning and memory problems, seizures, and psychoticdisorders, as well as disorders of vascular and muscle tone, breathing(asthma), hormone secretion, spermatocyte differentiation and motility,lymphocyte differentiation and cell proliferation.

Functionally, these BK beta subunits 2–4 are subunits of a Slo potassiumchannel. Such Slo potassium channels comprising the beta subunits maycontain one or more beta subunits along with alpha subunits from the Slofamily, e.g., Slo1, Slo2, or Slo3. The presence of the beta subunits ina Slo potassium channel modulates the activity of the channel and thusenhances channel diversity. For example, when a beta subunit associateswith other alpha monomers, the resulting channel may have an alteredsingle channel conductance as well as altered kinetic properties, e.g.,changes in activation or inactivation rates. Beta subunits have beenknown to increase the number of channels by helping the alpha subunitsget to the cell surface. BK beta subunits 2–4 also affect pharmacology,and change the sensitivity of the channel to natural ligands. Forexample, BK beta 3 increases the calcium sensitivity of Slo1 potassiumchannels and slowed activation kinetics, and BK beta 4 slows activationkinetics.

Structurally, each of the nucleotide sequences of BK beta subunits 2–4(e.g., SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6) encodes a polypeptidemonomer of a different size (SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5,respectively). The nucleotide sequence of BK beta 2 encodes apolypeptide monomer of approximately 257 amino acids with a predictedmolecular weight of approximately 29 kDa (SEQ ID NO:1) and a predictedrange of 24–34 kDa. The nucleotide sequence of BK beta 3 encodes apolypeptide monomer of approximately 210 amino acids with a predictedmolecular weight of approximately 23 kDa (SEQ ID NO:3) and a predictedrange of 18–28 kDa. The nucleotide sequence of BK beta 4 encodes apolypeptide monomer of approximately 235 amino acids with a predictedmolecular weight of approximately 27 kDa (SEQ ID NO:5) and a predictedrange of 22–32 kDa.

The present invention also provide polymorphic variants of BK betasubunits 2–4, as depicted in SEQ ID NO:1 for BK beta 2, SEQ ID NO:3 forBK beta 3, and SEQ ID NO:5 for BK beta 4, as follows.

For BK beta 2, in variant #1, a leucine residue is substituted for avaline residue at amino acid position 37; in variant #2, a leucineresidue is substituted for an isoleucine residue at amino acid position76; in variant #3, a valine residue is substituted for a isoleucineresidue at amino acid position 152; in variant #4, a isoleucine residueis substituted for a leucine residue at amino acid position 217.

For BK beta 3, in variant #1, a methionine residue is substituted for aisoleucine residue at amino acid position 25; in variant #2, a leucineresidue is substituted for a phenylalanine residue at amino acidposition 35; in variant #3, a lysine residue is substituted for anarginine residue at amino acid position 97; in variant #4, a methionineresidue is substituted for a isoleucine residue at amino acid position168.

For BK beta 4, in variant #1, a valine residue is substituted for anisoleucine residue at amino acid position 59; in variant #2, anasparagine residue is substituted for a glutamine residue at amino acidposition 83; in variant #3, a phenylalanine residue is substituted for aleucine residue at amino acid position 200; in variant #4, an isoleucineresidue is substituted for a valine residue at amino acid position 214.

Specific regions of BK beta subunits 2–4 nucleotide and amino acidsequence may be used to identify polymorphic variants, interspecieshomologs (orthologs), mutants, and alleles of BK beta subunits 2–4. Thisidentification can be made in vitro, e.g., under stringent hybridizationconditions and sequencing, or by using the sequence information in acomputer system for comparison with other nucleotide sequences.Typically, identification of polymorphic variants and alleles of BK betasubunits is made by comparing the amino acid sequence of the S1–S2region, which is comprised of the entire first two transmembranedomains, including the intervening extracellular domain. For BK beta 2,the S1–S2 region is approximately amino acids 39–209 (see, e.g., SEQ IDNO:1). For BK beta 3, the S1–S2 region is approximately amino acids20–191 (see, e.g., SEQ ID NO:3). For BK beta 4, the S1–S2 region isapproximately amino acids 50–217 (see, e.g., SEQ ID NO:5). Amino acididentity of about 70% or above, preferably 85%, more preferably 90–95%or above to either the S1–S2 region, or the entire BK beta subunitpolypeptide, typically demonstrates that a protein is a polymorphicvariant, interspecies homolog, or allele of a BK beta subunit. Sequencecomparison can be performed using any of the sequence comparisonalgorithms discussed below. Antibodies that bind specifically to a BKbeta subunit can also be used to identify alleles, interspecieshomologs, and mutants polymorphic variants.

Polymorphic variants, interspecies homologs, and alleles of BK betasubunits 2–4 are confirmed by co-expressing the putative BK beta subunitwith a Slo family alpha subunit, e.g., Slo1, Slo2, or Slo3, andexamining whether the beta subunit associates with a Slo potassiumchannel when co-expressed with a member of the Slo family. Functionalassays may be used to determine the characteristics of the Slo potassiumchannels formed in such ways. One assay is to determine changes incellular polarization by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques,e.g., “the cell-attached” mode, “the inside-out” mode, and “the wholecell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575–1595(1997)). These assays are used to demonstrate that a potassium channelcomprising a beta subunit having about 70% or greater, preferably 90% orgreater amino acid identity to the S1–S2 region, or the entire sequenceof a BK beta subunit such as BK beta 2, BK beta 3, or BK beta 4, is aspecies of a BK beta subunit 2, 3, or 4 because the subunit shares thesame functional characteristics. Typically, BK beta subunits having theamino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, areused as positive controls in comparison to the putative the BK betasubunit protein to demonstrate the identification of a polymorphicvariant or allele of a BK beta subunit.

BK beta subunit 2–4 nucleotide and amino acid sequence information mayalso be used to construct models of a Slo potassium channel in acomputer system. These models are subsequently used to identifycompounds that can activate or inhibit Slo potassium channels comprisingBK beta subunits 2, 3, or 4. Such compounds that modulate the activityof channels comprising BK beta subunits 2, 3, or 4 can be used toinvestigate the role of the BK beta subunits in modulation of channelactivity and in channel diversity.

The identification and cloning of BK beta subunits 2–4 for the firsttime provides a means for assaying for inhibitors and activators of Slopotassium channels that comprise BK beta subunits 2, 3, or 4.Biologically active BK beta subunits 2–4 are useful for testinginhibitors and activators of Slo potassium channels comprising a BK betasubunit and other Slo family members using in vivo and in vitroexpression that measure, e.g., changes in voltage or current. Suchactivators and inhibitors identified using a Slo potassium channelcomprising at least one BK beta subunit can be used to further study,e.g., regulation of the Slo family potassium channel, channel kineticsand conductance properties of channels. Such activators and inhibitorsare also useful as pharmaceutical agents for treating disease involvingabnormal ion flux, including diagnosis of CNS disorders such asmigraines, hearing and vision problems, learning and memory problems,seizures, and psychotic disorders, as well as disorders of vascular andmuscle tone, breathing (asthma), hormone secretion, spermatocytedifferentiation and motility, lymphocyte differentiation and cellproliferation.

Methods of detecting BK beta subunits 2–4 and expression of channelscomprising a BK beta subunit are also useful for diagnostic applicationsfor diseases involving abnormal ion flux, e.g., CNS disorders and otherdisorders described above. For example, chromosome localization of thegene encoding BK beta subunits 2–4 can be used to identify diseasescaused by and associated with the BK beta subunits. Methods of detectingBK beta subunits 2–4 are also useful for examining the role of BK betasubunits in Slo potassium channel diversity and modulation of Slopotassium channel activity.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “Slo family potassium channels” refers to a group of potassiumchannels composed of alpha subunits having a core ion channel motif of 6or 7 transmembrane domains and a pore domain (Butler et al., Science261:221–224 (1993); Meera et al., Proc. Natl. Acad. Sci. U.S.A.94:14066–71 (1997); Schreiber et al., J. Biol. Chem. 273:3509–16 (1998);Joiner et al., Nature Neurosci. 1:462–469 (1998)), and a long C-terminaldomain that appears to be involved in channel gating (Wei et al., Neuron13:671–681 (1994)). At least 3 members of this gene family exist inmammals: Slo1 (Butler et al., Science 261: 221–224 (1993); McCobb etal., Am. J. Physiol. 269:H767–H777 (1995)), Slo2 (Joiner et al., NatureNeurosci. 1:462–469 (1998)) and Slo3 (Schreiber et al., J. Biol. Chem.273:3509–16 (1998)). The Slo family channels have relatively largeconductance, ranging from 40–65 pS for Slo2 up to almost 300 pS forSlo1. Each Slo family channel is dually gated by voltage and a secondstimulus. For Slo1 and Slo2 that second stimulus is intracellularcalcium, and for Slo3 it is intracellular pH. The Slo family channelscan be identified on the basis of amino acid homology in a regionextending from the pore domain into the C-terminus, corresponding toamino acids 273–494 of the mouse Slo3 sequence (Schreiber et al., J.Biol. Chem. 273:3509–16 (1998)). In this region, each Slo family membershares at least 25% amino acid identity to other Slo family members inpairwise comparisons. Slo1 and Slo3 share more than 50% identity overthis region, whereas Slo2 is between 25–30% identical. No other channelsshare this level of identity with Slo channels over the entire length ofthis region (see FIG. 1).

The phrase “type II BK potassium channel” refers to a BK or Slo channelthat has slowed activation kinetics and resistance to charybdotoxinblock, e.g., Slo1/BK Beta 3 (see, e.g., Reinhart et al., Neuron2:1031–1041 (1989); see also FIG. 6).

The term “beta subunits” refers to polypeptide monomers that areauxiliary subunits of a potassium channel composed of alpha subunits;however, beta subunits alone cannot form a channel (see, e.g., U.S. Pat.No. 5,776,734). Beta subunits function, for example, to increase thenumber of channels by helping the alpha subunits reach the cell surface,change activation kinetics, and change the sensitivity of naturalligands binding to the channels. For example, BK beta 3 increases thecalcium sensitivity of Slo1 potassium channels and slowed activationkinetics, and BK beta 4 slows activation kinetics. The beta subunits canbe outside of the pore region and associated with alpha subunitscomprising the pore region. They can also contribute to the externalmouth of the pore region.

The term “transmembrane domain” refers to the region of the potassiumchannel subunit polypeptide that spans across the lipid bilayer membraneof the cells. Various families of the potassium channels have differentnumbers of transmembrane domains that travel across the cellularmembrane. Structurally, a transmembrane domain starts from the firstamino acid residue of the subunit sequence that enters into the cellularmembrane and ends with the last amino acid residue in the subunitsequence that leaves the cellular membrane.

“Homomeric” refers to a potassium channel composed of identical alphasubunits, while “heteromeric” refers to a potassium channel composed oftwo or more different types of alpha subunits. Both heteromeric andhomomeric channels can also include auxiliary beta subunits such as theBK beta subunits of the present invention.

The phrase “S1–S2 region” refers to the region of a BK beta subunit 2–4polypeptide comprising the entire first two transmembrane domains,including the intervening extracellular domain (approximately aminoacids 39–209 of BK beta 2, see SEQ ID NO:1; or amino acids 20–191 of BKbeta 3 see SEQ ID NO:3; or amino acids 50–217 of BK beta 4, see SEQ IDNO:5). This region is defined as the fragment from the first residue ofthe first transmembrane domain to the last residue of the secondtransmembrane domain. This region can be used to identify thepolymorphic variants, interspecies homologs, mutants, and alleles of BKbeta subunits.

The term “BK beta subunit” refers to a beta subunit of “BK” (largeconductance) potassium channel, also known as “Slo potassium channels.”The term “BK beta subunits 2–4” refers to BK beta 2, BK beta 3, and/orBK beta 4. A BK beta subunit is a polypeptide that is an auxiliarysubunit or monomer of a Slo family potassium channel. When a BK betasubunit is part of a Slo potassium channel, the channel has thecharacteristics of a Slo family channel, e.g., it is dually gated byvoltage and a second stimulus such as calcium or pH. BK beta subunitstherefore refer to polymorphic variants, alleles, mutants, andinterspecies homologs that: (1) have an S1–S2 or a full length codingregion that has greater than about 70% amino acid sequence identity,preferably about 80–90% amino acid sequence identity to S1–S2 or a fulllength sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; (2) bind topolyclonal antibodies raised against an immunogen comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:5, and conservatively modified variants thereof; (3)specifically hybridize under stringent hybridization conditions to asequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,or SEQ ID NO:6, and conservatively modified variants thereof; or (4) areamplified by primers that specifically hybridize under stringenthybridization conditions to the same sequence as a primers selected fromthe group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, and SEQ ID NO:1 (BK beta 2); or SEQ ID NO:12, SEQ ID NO:13 andSEQ ID NO:14 (BK beta 3); or SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17and SEQ ID NO:18 (BK beta 4).

The phrase “functional effects” in the context of assays for testingcompounds that modulate Slo channels comprising BK beta subunitsincludes the determination of any parameter that is indirectly ordirectly under the influence of the Slo channel comprising a BK betasubunit. It includes changes in ion flux, membrane potential, currentflow, transcription, ligand binding, G-protein binding, phosphorylationor dephosphorylation, signal transduction, receptor-ligand interactions,second messenger concentrations (e.g., cAMP, IP₃, or intracellularCa²⁺), as measured in vitro, in vivo, and ex vivo, and also includesother physiologic effects such increases or decreases ofneurotransmitter or hormone release.

By “determining the functional effect” is meant assays for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of BK beta subunit 2, 3, or 4. Such functionaleffects, e.g., physical and chemical effects, can be measured by anymeans known to those skilled in the art, e.g., patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, oocyte or tissue culture cell expression of BK betasubunits; transcriptional activation; ligand binding assays; voltage,membrane potential and conductance changes; ion flux assays; ligandbinding, hanges in intracellular second messengers such as cAMP andinositol triphosphate (IP₃); changes in intracellular calcium levels;neurotransmitter release, and the like.

“Inhibitors,” “activators,” and “modulators” of the Slo potassiumchannels comprising BK beta subunits 2, 3, or 4 refer to modulatormolecules identified using in vitro and in vivo assays for channelfunction. Inhibitors are compounds that decrease, block, prevent, delayactivation, inactivate, desensitize, or down regulate the channel.Activators are compounds that increase, open, activate, facilitate,enhance activation, sensitize or up regulate channel activity.Modulators include naturally occurring and synthetic ligands,antagonists, agonists, small chemical molecules and the like. Assays forinhibitors and activators include, e.g., co-expressing a BK beta subunitwith an alpha subunit in cells or cell membranes, applying putativemodulator compounds, and then measuring flux of ions through the Slochannel and determining the functional effect of the modulator asdescribed above.

Samples or assays comprising a Slo potassium channel comprising BK beta2, 3, or 4 that are treated with a potential activator or inhibitor arecompared to control samples without the inhibitor, to examine the extentof inhibition. Control samples (Slo channels comprising a BK betasubunit, untreated with inhibitors) are assigned a relative Slopotassium channel activity value of 100%. Inhibition of channelscomprising a BK beta subunit is achieved when the Slo potassium channelactivity value relative to the control is about 90%, preferably 50%,more preferably 25–0%. Activation of Slo channels comprising BK betasubunits is achieved when the Slo potassium channel activity valuerelative to the control is 110%, more preferably 150%, more preferably200–500% or 1000% and higher.

“Biologically active” BK beta subunit refers to BK beta subunits thatare part of a potassium channel having Slo family channel activity,tested as described above. Typically, such potassium channel contains atleast one type of alpha subunit, optionally more than one type of alphasubunit, and one or more BK beta subunits. Typically the potassiumchannels comprises at least one or two, preferably four alpha subunits,either identical or different.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolatednucleic acid encoding a BK beta subunit is separated from open readingframes that flank the BK beta subunit gene and encode proteins otherthan a BK beta subunit. The term “purified” denotes that a nucleic acidor protein gives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at least 85%pure, more preferably at least 95% pure, and most preferably at least99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group., e.g.,homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes (A, T,G, C, U, etc.).

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605–2608(1985); Rossolini et al., Mol. Cell. Probes 8:91–98 (1994)). Because ofthe degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon in an amino acidherein, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine, and TGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants and allelesof the invention.

The following groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Serine (S), Threonine (T);-   3) Aspartic acid (D), Glutamic acid (E);-   4) Asparagine (N), Glutamine (Q);-   5) Cysteine (C), Methionine (M);-   6) Arginine (R), Lysine (K), Histidine (H);-   7) Isoleucine (I), Leucine (L), Valine (V); and-   8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).    (see, e.g., Creighton, Proteins (1984) for a discussion of amino    acid properties).

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include 32P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins for which antisera or monoclonal antibodies are available(e.g., the polypeptide of SEQ ID NO:1 can be made detectable, e.g., byincorporating a radiolabel into the peptide, and used to detectantibodies specifically reactive with the peptide).

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence. A“labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides (i.e., 70%, 75%, 80%,85%, 90%, or 95% identity) that are the same, when compared and alignedfor maximum correspondence over a comparison window, as measured usingone of the following sequence comparison algorithms with the defaultparameters listed below, or by manual alignment and visual inspection.Such sequences are then said to be “substantially identical.” Thisdefinition also refers to the complement of a test sequence. Preferably,the percent identity exists over a region of the sequence that is atleast about 25 amino acids in length, more preferably over a region thatis 50 or 100 amino acids in length.

For sequence comparison, one sequence acts as a reference sequence, towhich test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are entered into a computer,subsequence coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. Default program parameterscan be used, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percent sequence identities forthe test sequences relative to the reference sequence, based on theprogram parameters.

A “comparison window,” as used herein, includes reference to a segmentof contiguous positions selected from the group consisting of from 20 to600, usually about 50 to about 200, more usually about 100 to about 150in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well-knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection.

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389–3402 (1977) and Altschul et al., J. Mol. Biol. 215:403–410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873–5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351–360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151–153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387–395 (1984).

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that twonucleotide sequences are substantially identical is that the sameprimers can be used to amplify both sequences.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5–10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, preferably 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with a wash in 0.2×SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50–70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′2, a dimer of Fab whichitself is a light chain joined to VH-CH1 by a disulfide bond. TheF(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3^(rd) ed. 1993).While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that such fragmentsmay be synthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495–497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77–96 in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc. (1985)). Techniques for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552–554(1990); Marks et al., Biotechnology 10:779–783 (1992)).

An “anti-BK beta subunit antibody” is an antibody or antibody fragmentthat specifically binds a polypeptide encoded by a BK beta subunit gene,cDNA, or a subsequence thereof.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to a BK beta subunit with the amino acid sequence of the subunitassociation region encoded in SEQ ID NO:1 can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withthe S1–S2 region of BK beta subunit 2 and not with other proteins,except for polymorphic variants and alleles of BK beta subunits. Thisselection may be achieved by subtracting out antibodies that cross reactwith molecules such as other BK beta subunits. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select antibodies specifically immunoreactive witha protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

The term “host cell” is meant that a cell that contains an expressionvector and supports the replication or expression of the expressionvector. Host cells may be prokaryotic cells such as E. coli, oreukaryotic cells such as yeast, insect, amphibian, or mammalian cellssuch as CHO, HeLa and the like, e.g., cultured cells, explants, andcells in vivo.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains a BK beta subunit or nucleic acid encoding a BK betasubunit protein. Such samples include, but are not limited to, tissueisolated from humans. Biological samples may also include sections oftissues such as frozen sections taken for histological purposes. Abiological sample is typically obtained from a eukaryotic organism,preferably eukaryotes such as fungi, plants, insects, protozoa, birds,fish, reptiles, and preferably a mammal such as rat, mice, cow, dog,guinea pig, or rabbit, and most preferably a primate such as chimpanzeesor humans.

III. Isolation of the Genes Encoding BK Beta Subunits

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Protein sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859–1862(1981), using an automated synthesizer, as described in Van Devanter etal., Nucleic Acids Res. 12:6159–6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137–149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21–26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences Encoding BKBeta Subunits

In general, the nucleic acid sequences encoding a beta subunit such asBK beta 2, BK beta 3, and BK beta 4, and related nucleic acid sequencehomologs are cloned from cDNA and genomic DNA libraries or isolatedusing amplification techniques with oligonucleotide primers. Forexample, BK beta 2 sequences are typically isolated from mouse or humannucleic acid (genomic or cDNA) by hybridizing with a nucleic acid probeor primer, the sequence of which can be derived from SEQ ID NO:2,preferably from the S1–S2 region of BK beta 2. Likewise, BK beta 3sequences are typically isolated from mouse or human nucleic acid(genomic or cDNA) by hybridizing with a nucleic acid probe or primer,the sequence of which can be derived from SEQ ID NO:4, preferably fromthe S1–S2 region of BK beta 3. BK beta 4 sequences are typicallyisolated from mouse or human nucleic acid (genomic or cDNA) byhybridizing with a nucleic acid probe or primer, the sequence of whichcan be derived from SEQ ID NO:6, preferably from the S1–S2 region of BKbeta 4.

Suitable tissues from which RNA and cDNA encoding BK beta subunits canbe isolated are as follows. For BK beta 2, the tissues include testis, Bcells, fetal heart, pregnant uterus, and melanocytes. BK beta 3 ishighly expressed in the brain. See Example II for a complete list of thetissues where BK beta 3 is expressed. For BK beta 4, heart and fetalkidney tissue are among the tissues from which it can be isolated. SeeExample III for a complete list of the tissues where BK beta 4 isexpressed.

Amplification techniques using primers can also be used to amplify andisolate BK beta subunits 2–4 from DNA or RNA. The following primers canbe used to amplify a sequence of BK beta 2, BK beta 3, or BK beta 4:

The following primers can be used to amplify a sequence of BK beta 2:

5-ATGACAGCCTTTCCTGCCTCAGGGAAG-3 (SEQ ID NO:7)5-AGATTTCTCTGCTCTTCCTTTGCTCCTCC-3 (SEQ ID NO:8)5-GGCTGGCTGGACTGTAGAAGCATG-3 (SEQ ID NO:9)5-GAGGCTGTCCAGATAAATCCCAAGTGC-3 (SEQ ID NO:10)5-GGACTGAGAAGCCCATCATGGCAAACC-3 (SEQ ID NO:11).Primers 7 and 8 can be used to amplify the entire coding region. Primers7 and 9 can be used to amplify the 5′ half, while primers 8 and 10 canbe used to amplify the 3′ half Primers 7 and 11 or 9 and 10 can be usedto amplify splice variants, as each set is in the same exon.

The following primers can be used to amplify a sequence of BK beta 3:

5-ATGGCGAAGCTCCGGGTGGCTTAC-3 (SEQ ID NO:12)5-TTAAGAGAACTTGCGCTTCTTCATGG-3 (SEQ ID NO:13)5-GATGTGCTTCTGCATCGCACTCATG-3 (SEQ ID NO:14).Primers 12 and 13 can be used to amplify the entire coding region andprimers 13 and 14 can be used to amplify splice variants, as they are inthe same exon.

The following primers can be used to amplify a sequence of BK beta 4:

5-AAGATGTCGATATGGACCAGTGGCC-3 (SEQ ID NO:15)5-TTATCTATTGATCCGTTGGATCCTCTC-3 (SEQ ID NO:16)5-CTCCTTCAGCTGTCCTCCAGACTGC-3 (SEQ ID NO:17)5-GTCCCAGTAGAATAGCTCGGTCCTC-3 (SEQ ID NO:18).Primers 15 and 16 can be used to amplify the entire coding region.Primers 15 and 18 can be used to amplify the 5′ half, and primers 16 and17 can be used to amplify the 3′ half.

These primers can be used, e.g., to amplify either the full lengthsequence or a probe of one to several hundred nucleotides, which is thenused to screen a human or mouse library for full-length BK beta subunitgenes.

Nucleic acids encoding BK beta subunits 2–4 can also be isolated fromexpression libraries using antibodies as probes. Such polyclonal ormonoclonal antibodies can be raised using the sequence of SEQ ID NO:1(for BK beta 2), SEQ ID NO:3 (for BK beta 3), and SEQ ID NO:5 (for BKbeta 4); or raised using the S1–S2 region of a BK beta subunit.

For BK beta subunits 2–4, polymorphic variants, alleles, andinterspecies homologs that are substantially identical to a BK betasubunit can be isolated using BK beta subunit nucleic acid probes andoligonucleotides under stringent hybridization conditions, by screeninglibraries. Alternatively, expression libraries can be used to clone eachof the BK beta subunits 2–4 and their polymorphic variants, alleles, andinterspecies homologs, by detecting expressed homologs immunologicallywith antisera or purified antibodies made against the S1–S2 region of BKbeta subunits 2–4, which also recognize and selectively bind to thehomolog.

To make a cDNA library, one should choose a source that is rich in mRNAof the particular BK beta subunit. For example, for BK beta 2, a cDNAlibrary from placenta, testis, or B cells is used. For BK beta 3, a cDNAlibrary from hippocampus is used. For BK beta 4, a cDNA library fromheart is used. For a more extensive list of tissues in which thesesubunits are expressed, see FIG. 2 and Example I. In each case, the mRNAis then made into cDNA using reverse transcriptase, ligated into arecombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. Methods for making and screeningcDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene25:263–269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA, preferably mouse or human, is extractedfrom the tissue and either mechanically sheared or enzymaticallydigested to yield fragments of about 12–20 kb. The fragments are thenseparated by gradient centrifugation from undesired sizes and areconstructed in bacteriophage lambda vectors. These vectors and phage arepackaged in vitro. Recombinant phage are analyzed by plaquehybridization as described in Benton & Davis, Science 196:180–182(1977). Colony hybridization is carried out as generally described inGrunstein et al., Proc. Natl. Acad. Sci. U.S.A., 72:3961–3965 (1975).

An alternative method of isolating the nucleic acid and the homologs ofBK beta subunits 2–4 combines the use of synthetic oligonucleotideprimers and amplification of an RNA or DNA template (see U.S. Pat. Nos.4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)). Methods such as polymerasechain reaction (PCR) and ligase chain reaction (LCR) can be used toamplify nucleic acid sequences of a BK beta subunit directly from mRNA,from cDNA, from genomic libraries or cDNA libraries. Degenerateoligonucleotides can be designed to amplify a BK beta subunit homologsusing the sequences provided herein. Restriction endonuclease sites canbe incorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of mRNAencoding a BK beta subunit in physiological samples, for nucleic acidsequencing, or for other purposes. Genes amplified by the PCR reactioncan be purified from agarose gels and cloned into an appropriate vector.

Gene expression of BK beta subunits 2–4 can also be analyzed bytechniques known in the art, e.g., reverse transcription andamplification of mRNA, isolation of total RNA or poly A+ RNA, northernblotting, dot blotting, in situ hybridization, RNase protection, probinghigh density oligonucleotide arrays, and the like.

Synthetic oligonucleotides can be used to construct recombinant genes ofBK beta subunits 2–4 for use as probes or for expression of protein.This method is performed using a series of overlapping oligonucleotidesusually 40–120 bp in length, representing both the sense and non-sensestrands of the gene. These DNA fragments are then annealed, ligated andcloned. Alternatively, amplification techniques can be used with preciseprimers to amplify a specific subsequence of a BK beta subunit gene. Thespecific subsequence is then ligated into an expression vector.

The genes for BK beta subunits 2–4 are typically cloned intointermediate vectors before transformation into prokaryotic oreukaryotic cells for replication and/or expression. These intermediatevectors are typically prokaryote vectors, e.g., plasmids, or shuttlevectors.

C. Expression of BK Beta Subunits 2–4 in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding BK beta subunits 2–4, one typically subclones a BK beta subunitgene into an expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing a BK beta subunit proteinare available in, e.g., E. col, Bacillus sp., and Salmonella (Palva etal., Gene 22:229–235 (1983); Mosbach et al., Nature 302:543–545 (1983).Kits for such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of nucleic acid encodinga BK beta subunit in host cells. A typical expression cassette thuscontains a promoter operably linked to the nucleic acid sequenceencoding a BK beta subunit and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. The nucleic acid sequence encoding BK betasubunit may be linked to a cleavable signal peptide sequence to promotesecretion of the encoded protein by the transformed cell, for isolationof individual subunits, e.g., for production of antibodies. Such signalpeptides would include, among others, the signal peptides from tissueplasminogen activator, insulin, and neuron growth factor, and juvenilehormone esterase of Heliothis virescens. Additional elements of thecassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplification,using genes such as such as thymidine kinase and dihydrofolatereductase. Alternatively, high yield expression systems not involvinggene amplification are also suitable, such as using a baculovirus vectorin insect cells, with sequence encoding BK beta subunits 2, 3, or 4under the direction of the polyhedrin promoter or other strongbaculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of a BK betasubunit, which is then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619–17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349–351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347–362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressing aBK beta subunit.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe BK beta subunit, which is recovered from the culture using standardtechniques identified below.

IV. Purification of BK Beta Subunits 2–4

Either naturally occurring or recombinant BK beta subunits 2–4 can bepurified for use in functional assays to identify modulators of Slochannels comprising BK beta subunits. Naturally occurring BK betasubunits 2–4 can be purified, e.g., from mouse or human tissue such astestis, heart, or hippocampus tissue and any other source of a BK betasubunit. Recombinant BK beta subunits 2–4 are purified from any suitableexpression system.

BK beta subunits 2–4 may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when a recombinant BK betasubunit 2–4 is being purified. For example, proteins having establishedmolecular adhesion properties can be reversible fused to a BK betasubunit. With the appropriate ligand, a BK beta subunit 2–4 can beselectively adsorbed to a purification column and then freed from thecolumn in a relatively pure form. The fused protein is then removed byenzymatic activity. Finally, a BK beta subunit 2–4 could be purifiedusing immunoaffinity columns.

A. Purification of BK Beta Subunits 2–4 from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of a BK betasubunit inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2–3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. The BK beta subunit isseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

Alternatively, it is possible to purify BK beta subunits 2–4 frombacteria periplasm. After lysis of the bacteria, when the BK betasubunit is exported into the periplasm of the bacteria, the periplasmicfraction of the bacteria can be isolated by cold osmotic shock inaddition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacterial cells arecentrifuged to form a pellet. The pellet is resuspended in a buffercontaining 20% sucrose. To lyse the cells, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes. The cell suspension is centrifugedand the supernatant decanted and saved. The recombinant proteins presentin the supernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

B. Standard Protein Separation Techniques for Purifying BK Beta Subunits2–4

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20–30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of a BK beta subunit can be used to isolated itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

A BK beta subunit can also be separated from other proteins on the basisof its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

V. Immunological Detection of BK Beta Subunits 2–4

In addition to the detection of BK beta subunits 2–4 genes and geneexpression using nucleic acid hybridization technology, one can also useimmunoassays to detect BK beta subunits 2–4. Immunoassays can be used toqualitatively or quantitatively analyze BK beta subunits 2–4. A generaloverview of the applicable technology can be found in Harlow & Lane,Antibodies: A Laboratory Manual (1988).

A. Antibodies to BK Beta Subunits 2–4

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with BK beta subunits 2–4 are known to those of skill inthe art (see, e.g., Coligan, Current Protocols in Immunology (1991);Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles andPractice (2d ed. 1986); and Kohler & Milstein, Nature 256:495–497(1975). Such techniques include antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science246:1275–1281 (1989); Ward et al., Nature 341:544–546 (1989)).

A number of immunogens comprising BK beta subunits 2–4 may be used toproduce antibodies specifically reactive with the BK beta subunit. Forexample, recombinant BK beta subunit, BK beta 2, BK beta 3, or BK beta4, or a antigenic fragment thereof such as the S1–S2 region, is isolatedas described herein. Recombinant protein can be expressed in eukaryoticor prokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511–519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275–1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-beta subunitproteins or even other related proteins from other organisms (e.g.,other BK beta subunits), using a competitive binding immunoassay.Specific polyclonal antisera and monoclonal antibodies will usually bindwith a K_(d) of at least about 0.1 mM, more usually at least about 1 μM,preferably at least about 0.1 μM or better, and most preferably, 0.01 μMor better.

Once the specific antibodies against a BK beta subunit 2–4 areavailable, a BK beta subunit 2–4 can be detected by a variety ofimmunoassay methods. For a review of immunological and immunoassayprocedures, see Basic and Clinical Immunology (Stites & Terr eds.,7^(th) ed. 1991). Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &Lane, supra.

B. Immunological Binding Assays

BK beta subunits 2–4 can be detected and/or quantified using any of anumber of well recognized immunological binding assays (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a reviewof the general immunoassays, see also Methods in Cell Biology:Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic andClinical Immunology (Stites & Terr, eds., 7^(th) ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this caseBK beta 2, BK beta 3, or BK beta 4, or an antigenic subsequencethereof). The antibody (e.g., anti-BK beta subunits 2, 3, or 4) may beproduced by any of a number of means well known to those of skill in theart and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled the BK beta subunitor a labeled anti-BK beta subunit antibody. Alternatively, the labelingagent may be a third moiety, such a secondary antibody, thatspecifically binds to the antibody/BK beta 2–4 subunit complex (asecondary antibody is typically specific to antibodies of the speciesfrom which the first antibody is derived). Other proteins capable ofspecifically binding immunoglobulin constant regions, such as protein Aor protein G may also be used as the label agent. These proteins exhibita strong non-immunogenic reactivity with immunoglobulin constant regionsfrom a variety of species (see, e.g., Kronval et al., J. Immunol.111:1401–1406 (1973); Akerstrom et al., J. Immunol. 135:2589–2542(1985)). The labeling agent can be modified with a detectable moiety,such as biotin, to which another molecule can specifically bind, such asstreptavidin. A variety of detectable moieties are well known to thoseskilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting BK beta subunits in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, an anti-BK beta subunit 2, 3, or 4antibody can be bound directly to a solid substrate on which they areimmobilized. These immobilized antibodies then capture the BK betasubunit present in the test sample. The BK beta subunit is thusimmobilized and then bound by a labeling agent, such as a second BK betasubunit antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second or third antibody is typically modified with adetectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive Assay Formats

In competitive assays, the amount of the BK beta subunit present in thesample is measured indirectly by measuring the amount of a known, added(exogenous) BK beta subunit displaced (competed away) from an anti-BKbeta subunit antibody by the unknown BK beta subunit present in asample. In one competitive assay, a known amount of the BK beta subunitis added to a sample and the sample is then contacted with an antibodythat specifically binds to the BK beta subunit. The amount of exogenousBK beta subunit bound to the antibody is inversely proportional to theconcentration of the BK beta subunit present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of BK beta subunit bound to the antibody maybe determined either by measuring the amount of BK beta subunit presentin a BK beta subunit/antibody complex, or alternatively by measuring theamount of remaining uncomplexed protein. The amount of BK beta subunitmay be detected by providing a labeled BK beta subunit molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay a known BK beta subunit such as BK beta 3 is immobilized on asolid substrate. A known amount of anti-BK beta 3 antibody is added tothe sample, and the sample is then contacted with the immobilized BKbeta 3. The amount of anti-BK beta 3 antibody bound to the knownimmobilized BK beta 3 is inversely proportional to the amount of BK beta3 present in the sample. Again, the amount of immobilized antibody maybe detected by detecting either the immobilized fraction of antibody orthe fraction of the antibody that remains in solution. Detection may bedirect where the antibody is labeled or indirect by the subsequentaddition of a labeled moiety that specifically binds to the antibody asdescribed above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations for a BK beta subunit. 2, 3, or 4. Forexample, a protein at least partially encoded by SEQ ID NO:1, preferablyincluding S1–S2 region, can be immobilized to a solid support. Otherproteins, preferably other BK beta subunits such as BK beta 1, BK beta3, or BK beta 4, are added to the assay so as to compete for binding ofthe antisera to the immobilized antigen. The ability of the addedproteins to compete for binding of the antisera to the immobilizedprotein is compared to the ability of the BK beta subunit encoded by SEQID NO:1 compete with itself. The percent crossreactivity for the aboveproteins is calculated, using standard calculations. Those antisera withless than 10% crossreactivity with each of the added proteins listedabove are selected and pooled. The cross-reacting antibodies areoptionally removed from the pooled antisera by immunoabsorption with theadded considered proteins, e.g., distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of BK beta 2, tothe immunogen protein. In order to make this comparison, the twoproteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required to inhibit 50% of binding is less than 10 timesthe amount of the protein encoded by SEQ ID NO:1, or SEQ ID NO:3, or SEQID NO:5 that is required to inhibit 50% of binding, then the secondprotein is said to specifically bind to the polyclonal antibodiesgenerated to the respective BK beta subunit immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of the BK beta subunit in the sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind the BK beta subunit. The anti-BK beta subunitantibodies specifically bind to the BK beta subunit on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to the anti-BK betasubunit antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34–41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize the BK betasubunit, or secondary antibodies that recognize anti-BK beta subunitantibodies.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of Slo Channels Comprising BK Beta Subunits2–4

A. Assays

BK beta subunits 2–4 and their alleles, interspecies homologs,polymorphic variants are auxiliary subunits of Slo family potassiumchannels. The activity of a Slo potassium channel comprising a BK betasubunit or monomer can be assessed using a variety of in vitro and invivo assays, e.g., measuring voltage, current, measuring membranepotential, measuring ion flux, e.g., potassium or rubidium, measuringpotassium concentration, measuring second messengers and transcriptionlevels, measuring ligand binding, and using e.g., voltage-sensitivedyes, radioactive tracers, and patch-clamp electrophysiology.

Furthermore, such assays can be used to test for inhibitors andactivators of channels comprising a BK beta subunit. Such modulatorsthat target specific Slo channels differing in BK beta subunitscomposition are useful for treating a variety of diseases that alterexcitation and secretion, such as CNS disorders such as migraines,hearing and vision problems, learning and memory problems, seizures,psychotic disorders, and as neuroprotective agents (e.g., to preventstroke), and disorders of vascular and muscle tone, breathing (asthma),hormone secretion, spermatocyte differentiation and motility, lymphocytedifferentiation and cell proliferation. These modulators are usefulbecause they provide target specificity based on the expression pattersof BK beta subunits. For example, modulators targeting Slo channelscomprising BK beta 3 subunits, which are highly expressed in the CNS,are useful in treating excitatory and secretory disorders of the CNS.Modulators of Slo channels comprising BK beta 2 subunits would be usefulin treating diseases related to glandular secretion and immune celldifferentiation and proliferation. Such modulators are also useful forinvestigation of the channel diversity provided by BK beta subunits 2–4and the regulation/modulation of channel activity provided by BK betasubunits 2–4.

Modulators of the Slo potassium channels are tested using biologicallyactive BK beta subunits 2–4, either recombinant or naturally occurring.The BK beta subunit or a channel comprising such a subunit can beisolated, expressed in a cell, or expressed in a membrane derived from acell. In such assays, each of the BK beta subunits is typicallyco-expressed with an alpha subunit of the Slo family such as Slo 1, Slo2, or Slo 3. Modulation is tested using one of the in vitro or in vivoassays described above. Samples or assays that are treated with apotential Slo potassium channel inhibitor or activator are compared tocontrol samples without the test compound, to examine the extent ofmodulation. Control samples (A Slo channel comprising a BK beta subunit,untreated with activators or inhibitors) are assigned a relative Slopotassium channel activity value of 100. Inhibition of channelscomprising a BK beta subunit 2, 3, or 4 is achieved when the Slopotassium channel activity value relative to the control is about 90%,preferably 50%, more preferably 25%. Activation of channels comprising aBK beta subunit is achieved when the Slo potassium channel activityvalue relative to the control is 110%, more preferably 150%, morepreferable 200% higher. Compounds that increase open probability of achannel comprising BK beta subunit 2, 3, or 4, by decreasing theprobability of it being closed, increasing conductance through thechannel, and allowing the passage of ions.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing the Slo potassium channel comprising a BK beta subunit 2, 3,or 4. A preferred means to determine changes in cellular polarization isby measuring changes in current (thereby measuring changes inpolarization) with voltage-clamp and patch-clamp techniques, e.g., the“cell-attached” mode, the “inside-out” mode, and the “whole cell” mode(see, e.g., Ackerman et al., New Engl. J. Med. 336:1575–1595 (1997)).Whole cell currents are conveniently determined using the standardmethodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85 (1981).Other known assays include: radiolabeled rubidium flux assays andfluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67–75 (1988); Daniel etal., J. Pharmacol. Meth. 25:185–193 (1991); Holevinsky et al., J.Membrane Biology 137:59–70 (1994)). Assays for compounds capable ofinhibiting or increasing potassium flux through the channel proteinscomprising a BK beta subunit 2, 3, or 4 can be performed by applicationof the compounds to a bath solution in contact with and comprising cellshaving an channel of the present invention (see, e.g., Blatz et al.,Nature 323:718–720 (1986); Park, J. Physiol. 481:555–570 (1994)).Generally, the compounds to be tested are present in the range from 1 pMto 100 mM.

The effects of the test compounds upon the function of the channels canbe measured by changes in the electrical currents or ionic flux or bythe consequences of changes in currents and flux. Changes in electricalcurrent or ionic flux are measured by either increases or decreases influx of cations such as potassium or rubidium ions. The cations can bemeasured in a variety of standard ways. They can be measured directly byconcentration changes of the ions or indirectly by membrane potential orby radiolabeling of the ions. Consequences of the test compound on ionflux can be quite varied. Accordingly, any suitable physiological changecan be used to assess the influence of a test compound on the channelsof this invention. The effects of a test compound can be measured by atoxin binding assay. When the functional consequences are determinedusing intact cells or animals, one can also measure a variety of effectssuch as transmitter release (e.g., dopamine), hormone release (e.g.,insulin), transcriptional changes to both known and uncharacterizedgenetic markers (e.g., northern blots), cell volume changes (e.g., inred blood cells), immunoresponses (e.g., T cell activation), changes incell metabolism such as cell growth or pH changes, and changes inintracellular second messengers such as [Ca²⁺].

Preferably, a BK beta subunit 2, 3, or 4 that is a part of the Slopotassium channel used in the assay will be selected from a subunithaving a sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 or aconservatively modified variant thereof. Alternatively, the BK betasubunit of the assay will be derived from a eukaryote and include anamino acid subsequence having amino acid sequence identity to the BKbeta subunit. Generally, the amino acid sequence identity will be atleast about 70%, preferably at least 80%, most preferably at least90–95%. Alternatively, the BK beta subunit of the assay is derived froma eukaryote and include an amino acid subsequence having amino acidsequence identity to the S1–S2 region of the BK beta subunit. Generally,the amino acid sequence identity will be at least 70%, preferably atleast 80%, and most preferably at least 90–95%.

BK beta subunit 2, 3, or 4 polymorphic variants, alleles, andinterspecies homologs will generally confer substantially similarproperties on a channel, e.g., modulating the activity of Slo channelscomprising such a subunit, as described above. Furthermore, Slo familypotassium channels comprising such putative BK beta subunit homologsshould respond to modulatory compounds in a manner similar to Slopotassium channels comprising BK beta 2, 3, or 4. In one embodiment, acell or cell membrane in which has been expressed a putative BK betahomolog, allele, or interspecies homolog is assayed with a compound thatmodulates Slo family potassium channels. In one embodiment, compoundswith known modulatory activity for Slo family potassium channelscomprising known BK beta subunits are used in assays to identifyputative BK beta subunit homologs.

B. Modulators

The compounds tested as modulators of Slo channels comprising a BK betasubunit can be any small chemical compound, or a biological entity, suchas a protein, sugar, nucleic acid or lipid. Alternatively, modulatorscan be genetically altered versions of a BK beta subunit. Typically,test compounds will be small chemical molecules and peptides.Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487–493(1991) and Houghton et al., Nature 354:84–88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909–6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217–9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309–314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520–1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressinga Slo channel comprising a BK beta subunit is attached to a solid phasesubstrate. In the high throughput assays of the invention, it ispossible to screen up to several thousand different modulators orligands in a single day. In particular, each well of a microtiter platecan be used to run a separate assay against a selected potentialmodulator, or, if concentration or incubation time effects are to beobserved, every 5–10 wells can test a single modulator. Thus, a singlestandard microtiter plate can assay about 100 (e.g., 96) modulators. If1536 well plates are used, then a single plate can easily assay fromabout 100–about 1500 different compounds. It is possible to assayseveral different plates per day; assay screens for up to about6,000–20,000 different compounds is possible using the integratedsystems of the invention.

VII. Computer Assisted Drug Design Using BK Beta Subunits 2–4

Yet another assay for compounds that modulate channels comprising BKbeta subunits 2, 3, or 4 involves computer assisted drug design, inwhich a computer system is used to generate a three-dimensionalstructure of the BK subunit based on the structural information encodedby the amino acid sequence. The amino acid sequence entered into thesystem interacts directly and actively with a preestablished algorithmin a computer program to yield secondary, tertiary, and quaternarystructural models of the protein. The models of the protein structureare then examined to identify regions of the structure that have theability to bind, e.g., ligands or other potassium channel subunits.These regions are then used to identify ligands that bind to the proteinor region where the BK beta subunit interacts with other potassiumchannel subunits. Information generated by the computer system, e.g.,structures, sequence comparisons and the like, are saved on computerreadable substrates.

The three-dimensional structural model of the protein is generated byentering channel protein amino acid sequences of at least 10 amino acidresidues or corresponding nucleic acid sequences encoding a BK betasubunit 2, 3, or 4 into the computer system. The amino acid sequence ofeach of the monomers is selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5, for BK beta 2, BK beta 3, and BKbeta 4, respectively, and a conservatively modified versions thereof.The amino acid sequence represents the primary sequence or subsequenceof each of the proteins, which encodes the structural information of theprotein. At least 10 residues of the amino acid sequence (or anucleotide sequence encoding 10 amino acids) are entered into thecomputer system from computer keyboards, computer readable substratesthat include, but are not limited to, electronic storage media (e.g.,magnetic diskettes, tapes, cartridges, and chips), optical media (e.g.,CD ROM), information distributed by internet sites, and by RAM. Thethree-dimensional structural model of the channel protein is thengenerated by the interaction of the amino acid sequence and the computersystem, using software known to those of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the monomer and the potassium channel protein comprisingmultiple monomers. The software looks at certain parameters encoded bythe primary sequence to generate the structural model. These parametersare referred to as “energy terms,” and primarily include electrostaticpotentials, hydrophobic potentials, solvent accessible surfaces, andhydrogen bonding. Secondary energy terms include van der Waalspotentials. Biological molecules form the structures that minimize theenergy terms in a cumulative fashion. The computer program is thereforeusing these terms encoded by the primary structure or amino acidsequence to create the secondary structural model.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the BK beta subunit protein to identify ligands that bind to theBK beta subunit. Binding affinity between the protein and ligands isdetermined using energy terms to determine which ligands have anenhanced probability of binding to the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of BK beta subunit genes.Such mutations can be associated with disease states. Once the variantsare identified, diagnostic assays can be used to identify patientshaving such mutated genes associated with disease states. Identificationof the mutated BK beta subunit genes involves receiving input of a firstnucleic acid, selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4, and SEQ ID NO:6, or an amino acid sequence encoding the BK betasubunit, selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5, and a conservatively modified versions thereof. Thesequence is entered into the computer system as described above. Thefirst nucleic acid or amino acid sequence is then compared to a secondnucleic acid or amino acid sequence that has substantial identity to thefirst sequence. The second sequence is entered into the computer systemin the manner described above. Once the first and second sequences arecompared, nucleotide or amino acid differences between the sequences areidentified. Such sequences can represent allelic differences in the BKbeta subunit genes, and mutations associated with disease states.

BK beta subunits 2–4 and the Slo potassium channels comprising these BKbeta subunits can be used with high density oligonucleotide arraytechnology (e.g., GeneChip™) to identify homologs and polymorphicvariants of the BK beta subunits in this invention. In the case wherethe homologs being identified are linked to a known disease, they can beused with high density oligonucleotide array technology as a diagnostictool in detecting the disease in a biological sample (see, e.g.,Gunthand et al., AIDS Res. Hum. Retroviruses 14: 869–876 (1998); Kozalet al., Nat. Med. 2:753–759 (1996); Matson et al., Anal. Biochem.224:110–106 (1995); Lockhart et al., Nat. Biotechnol. 14:1675–1680(1996); Gingeras et al., Genome Res. 8:435–448 (1998); Hacia et al.,Nucleic Acids Res. 26:3865–3866 (1998)).

VIII. Cellular Transfection and Gene Therapy

The present invention provides the nucleic acids of BK beta subunits 2–4for the transfection of cells in vitro and in vivo. These nucleic acidscan be inserted into any of a number of well known vectors for thetransfection of target cells and organisms as described below. Thenucleic acids are transfected into cells, ex vivo or in vivo, throughthe interaction of the vector and the target cell. The nucleic acidsencoding BK beta subunits 2, 3, or 4, under the control of a promoter,then expresses a BK beta subunit of the present invention, therebymitigating the effects of absent, partial inactivation, or abnormalexpression of the BK beta subunit gene.

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and viral infection in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science256:808–813 (1992); Nabel & Felgner, TIBTECH 11:211–217 (1993); Mitani &Caskey, TIBTECH 11:162–166 (1993); Mulligan, Science 926–932 (1993);Dillon, TIBTECH 11:167–175 (1993); Miller, Nature 357:455–460 (1992);Van Brunt, Biotechnology 6(10):1149–1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8:35–36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31–44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Böhm eds., 1995); and Yu etal., Gene Therapy 1:13–26 (1994)).

Delivery of the gene or genetic material into the cell is the firstcritical step in gene therapy treatment of disease. A large number ofdelivery methods are well known to those of skill in the art.Preferably, the nucleic acids are administered for in vivo or ex vivogene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808–813 (1992); Nabel & Felgner, TIBTECH11:211–217 (1993); Mitani & Caskey, TIBTECH 11:162–166 (1993); Dillon,TIBTECH 11:167–175 (1993); Miller, Nature 357:455–460 (1992); Van Brunt,Biotechnology 6(10):1149–1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35–36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31–44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13–26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described in,e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat.No. 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404–410 (1995); Blaese etal., Cancer Gene Ther. 2:291–297 (1995); Behr et al., Bioconjugate Chem.5:382–389 (1994); Remy et al., Bioconjugate Chem. 5:647–654 (1994); Gaoet al., Gene Therapy 2:710–722 (1995); Ahmad et al., Cancer Res.52:4817–4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6–10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731–2739 (1992); Johann et al., J. Virol. 66:1635–1640 (1992);Sommerfelt et al., Virol. 176:58–59 (1990); Wilson et al., J. Virol.63:2374–2378 (1989); Miller et al., J. Virol. 65:2220–2224 (1991);PCT/US94/05700).

In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38–47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793–801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251–3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072–2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466–6470 (1984); andSamulski et al., J. Virol. 63:03822–3828 (1989).

In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048–305 (1995); Kohn etal., Nat. Med. 1: 1017–102 (1995); Malech et al., Proc. Natl. Acad. Sci.U.S.A. 94:22 12133–12138 (1997)). PA317/pLASN was the first therapeuticvector used in a gene therapy trial. (Blaese et al., Science 270:475–480(1995)). Transduction efficiencies of 50% or greater have been observedfor MFG-S packaged vectors. (Ellem et al., Immunol Immunother.44(1):10–20 (1997); Dranoff et al., Hum. Gene Ther. 1:111–2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702–3 (1998), Kearns et al., Gene Ther.9:748–55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used transient expression gene therapy, because they canbe produced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiply types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083–9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 24:1 5–10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083–1089 (1998); Welsh et al., Hum. Gene Ther. 2:205–18(1995); Alvarez et al., Hum. Gene Ther. 5:597–613 (1997); Topfet al.,Gene Ther. 5:507–513 (1998); Sterman et al., Hum. Gene Ther. 7:1083–1089(1998).

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et al., Proc. Natl. Acad. Sci. U.S.A.92:9747–9751 (1995), reported that Moloney murine leukemia virus can bemodified to express human heregulin fused to gp70, and the recombinantvirus infects certain human breast cancer cells expressing humanepidermal growth factor receptor. This principle can be extended toother pairs of virus expressing a ligand fusion protein and target cellexpressing a receptor. For example, filamentous phage can be engineeredto display antibody fragments (e.g., FAB or Fv) having specific bindingaffinity for virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to nonviral vectors. Such vectors can be engineered tocontain specific uptake sequences thought to favor uptake by specifictarget cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a nucleicacid (gene or cDNA), and re-infused back into the subject organism(e.g., patient). Various cell types suitable for ex vivo transfectionare well known to those of skill in the art (see, e.g., Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The nucleicacids are administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch nucleic acids are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

IX. Pharmaceutical Compositions

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed., 1989).

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, usuallysucrose and acacia or tragacanth, as well as pastilles comprising theactive ingredient in an inert base, such as gelatin and glycerin orsucrose and acacia emulsions, gels, and the like containing, in additionto the active ingredient, carriers known in the art.

The nucleic acids, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid as described above in thecontext of ex vivo therapy can also be administered intravenously orparenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of the Slo family potassium channel comprising a BKbeta subunit, the physician evaluates circulating plasma levels of thevector, vector toxicities, progression of the disease, and theproduction of anti-vector antibodies. In general, the dose equivalent ofa naked nucleic acid from a vector is from about 1 μg to 100 μg for atypical 70 kilogram patient, and doses of vectors which include aretroviral particle are calculated to yield an equivalent amount oftherapeutic nucleic acid.

For administration, inhibitors and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

X. Kits

BK beta subunits 2–4 and their homologs are useful tools for examiningexpression and regulation of Slo family potassium channels. Reagentsthat specifically hybridize to nucleic acids encoding BK beta subunit 2,3, or 4, such as BK beta subunits 2–4 probes and primers, and reagentsthat specifically bind to BK beta subunits 2–4 protein, e.g.,antibodies, are used to examine expression and regulation.

Nucleic acid assays for the presence of BK beta subunits 2–4 DNA and RNAin a sample include numerous techniques are known to those skilled inthe art, such as Southern analysis, northern analysis, dot blots, RNaseprotection, S1 analysis, amplification techniques such as PCR and LCR,high density oligonucleotide array analysis, and in situ hybridization.In in situ hybridization, for example, the target nucleic acid isliberated from its cellular surroundings in such as to be available forhybridization within the cell while preserving the cellular morphologyfor subsequent interpretation and analysis. The following articlesprovide an overview of the art of in situ hybridization: Singer et al.,Biotechniques 4:230–250 (1986); Haase et al., Methods in Virology, vol.VII, pp. 189–226 (1984); and Nucleic Acid Hybridization: A PracticalApproach (Hames et al., eds. 1987). In addition, a BK beta subunit canbe detected with the various immunoassay techniques described above. Thetest sample is typically compared to both a positive control (e.g., asample expressing recombinant BK beta subunit 2, 3, or 4) and a negativecontrol.

The present invention also provides for kits for screening modulators ofthe potassium channels. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: BK beta subunits 2, 3, and/or 4,reaction tubes, and instructions for testing the activities of Slopotassium channels comprising a BK beta subunit. A wide variety of kitsand components can be prepared according to the present invention,depending upon the intended user of the kit and the particular needs ofthe user. For example, the kit can be tailored for in vitro or in vivoassays for measuring the activity of a Slo family potassium channelcomprising a BK beta subunit.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Isolation of Nucleic Acids Encoding BK Beta 2 and FunctionalAnalysis of Potassium Channels Containing BK Beta 2

Using PCR and primers, according to standard conditions, BK beta 2 isamplified from a human testis cDNA library. The following primers areused for amplification of BK beta 2:

(1) 5-ATGACAGCCTTTCCTGCCTCAGGGAAG-3 (SEQ ID NO:7) (2)5-AGATTTCTCTGCTCTTCCTTTGCTCCTCC-3 (SEQ ID NO:8) (3)5-GGCTGGCTGGACTGTAGAAGCATG-3 (SEQ ID NO:9) (4)5-GAGGCTGTCCAGATAAATCCCAAGTGC-3 (SEQ ID NO:10) (5)5-GGACTGAGAAGCCCATCATGGCAAACC-3 (SEQ ID NO:11)

Primers (1) and (2) are used together to amplify the entire codingsequence. Primers (1) and (3) are used to amplify the 5′ half of thecoding sequence, while primers (2) and (4) are used to amplify the 3′half. Primers (1) and (5) are used as a set of oligos that may amplifyany splice variants since they are in the same exon. Primers (3) and (4)might also be good choices for this purpose.

PCR conditions are as follows: 95 degrees for 30 seconds, 68–58 degreesfor 15 seconds, 72 degrees for 45 seconds. The reaction is run for 40cycles.

The PCR products are subcloned into plasmids and sequenced according tostandard techniques. The nucleotide and amino acid sequences of BK beta2 are provided, respectively, in SEQ ID NO:2 and SEQ ID NO:1.

BK beta 2 monomer is co-expressed with a Slo alpha subunit monomeraccording to standard methodology to demonstrate its ability toassociate with potassium channels made by alpha subunits the Slopotassium channel family. Changes in Slo potassium channel activity areobserved by measuring their current/voltage relationship at givencalcium concentrations.

BK beta 2 expression patterns were analyzed using northern blots andmRNA dot blots (FIG. 2). BK beta expression was high in placenta, whilelower levels of expression were detected in a variety of tissuesincluding substantia nigra, spinal cord, lung, prostate, testis,pancreas, stomach, small intestine, pituitary gland, adrenal gland,thyroid, salivary gland, mammary gland, spleen, thymus, lymph node, bonemarrow, fetal liver, fetal spleen, and fetal lung. Trace levels ofexpress were detected in a variety of nervous and peripheral tissues(FIG. 2).

Example II Isolation of Nucleic Acids Encoding BK Beta 3 and FunctionalAnalysis of Potassium Channels Containing BK Beta 3

Using PCR and primers, according to standard conditions, BK beta 3 isamplified from a human hippocampus cDNA library. The following primersare used for amplification of BK beta 3:

(1) 5-ATGGCGAAGCTCCGGGTGGCTTAC-3 (SEQ ID NO:12) (2)5-TTAAGAGAACTTGCGCTTCTTCATGG-3 (SEQ ID NO:13) (3)5-GATGTGCTTCTGCATCGCACTCATG-3 (SEQ ID NO:14)

Primers (1) and (2) are used together to amplify the entire codingsequence. Primers (2) and (3) are used to amplify the 3′ end of thecoding sequence. They are in the same exon.

PCR conditions are as follows: 95 degrees for 30 seconds, 68–58 degreesfor 15 seconds, 72 degrees for 45 seconds. The reaction is run for 40cycles.

The PCR products are subcloned into plasmids and sequenced according tostandard techniques. The nucleotide and amino acid sequences of BK beta3 are provided, respectively, in SEQ ID NO:4 and SEQ ID NO:3.

BK beta 3 monomer is co-expressed with a Slo alpha subunit monomeraccording to standard methodology to demonstrate its ability toassociate with potassium channels made by alpha subunits the Slopotassium channel family. Changes in Slo potassium channel activity areobserved by measuring their current/voltage relationship at givencalcium concentrations.

BK beta 3 expression patterns were analyzed using northern blots andmRNA dot blots (FIG. 2). It is highly expressed in the brain and wasexpressed in all neuronal tissues that were tested. Its expression wasespecially high in the hippocampus, putamen, substantia nigra, temporallobe, thalamus, nucleus accumbens, cerebral cortex, amygdala, occipitallobe and fetal brain, with lower expression levels in the cerebellum andmedulla.

BK beta 3 was also expressed at moderate to low levels in severalperipheral tissues, such as adrenal gland, fetal heart, fetal lung,small intestine, fetal kidney, appendix, lung, placenta, thyroid, ovary,pituitary, and prostate. There were trace levels of expression in heart,aorta, colon, bladder, uterus, stomach, testis, salivary gland, kidney,thymus, and spleen.

When co-expressed with human Slo, BK beta 3 causes a hyperpolarizedshift in the activation curve vs. voltage curve when compared to Slo1expressed alone (FIG. 3). Furthermore, when coexpressed with Slo1, BKbeta 3 slows the activation of Slo channels and causes a smallhyperpolarized shift in the activation vs. voltage curve that is mostpronounced at higher calcium concentrations (see FIGS. 4–5). Inaddition, BK beta 3 greatly reduces the sensitivity of Slo1 channels tocharybdotoxin (FIG. 6). When expressed alone, Slo1 channels are almostcompletely blocked by 100 nM charybdotoxin. However, when expressed withBK beta 3, the Slo1 current is virtually unblocked by 100 nMcharybdotoxin. The Slo1/BKB3 channels have similar properties to theType II BK channels that have been identified in mammalian brain(Reinhart et al., Neuron 2:1031–1041 (1989)), which also have slowedactivation kinetics and resistance to charybdotoxin block. It istherefore likely that modulators of mammalian type II BK channels can beidentified by screening for compounds that modulate heterologouslyexpressed channels consisting of Slo1 and BK beta 3 subunits.

Example III Isolation of Nucleic Acids Encoding BK Beta 4 and FunctionalAnalysis of Potassium Channels Containing BK Beta 4

Using PCR and primers, according to standard conditions, BK beta 4 isamplified from a human heart cDNA library. The following primers areused for amplification of BK beta 4:

(1) 5-AAGATGTCGATATGGACCAGTGGCC-3 (SEQ ID NO:15) (2)5-TTATCTATTGATCCGTTGGATCCTCTC-3 (SEQ ID NO:16) (3)5-CTCCTTCAGCTGTCCTCCAGACTGC-3 (SEQ ID NO:17) (4)5-GTCCCAGTAGAATAGCTCGGTCCTC-3 (SEQ ID NO:18)

Primers (1) and (2) are used together to amplify the entire codingsequence. Primers (1) and (4) are used to amplify the 5′ end of thecoding sequence. Primers (2) and (3) are used to amplify the 3′ half ofcoding.

PCR conditions are as follows: 95 degrees for 30 seconds, 68–58 degreesfor 15 seconds, 72 degrees for 45 seconds. The reaction is run for 40cycles.

The PCR products are subcloned into plasmids and sequenced according tostandard techniques. The nucleotide and amino acid sequences of BK beta4 are provided, respectively, in SEQ ID NO:6 and SEQ ID NO:5.

BK beta 4 monomer is co-expressed with a Slo alpha subunit monomeraccording to standard methodology to demonstrate its ability toassociate with potassium channels made by alpha subunits the Slopotassium channel family. Changes in Slo potassium channel activity areobserved by measuring their current/voltage relationship at givencalcium concentrations.

BK beta 4 expression patterns were analyzed using northern blots andmRNA dot blots (FIG. 2). The highest expression of BK beta 4 was foundin fetal kidney. Moderate levels of expression were also present inovary and pituitary gland. Low levels of expression were found in fetallung, heart, fetal brain, occipital lobe, substantia nigra, thalamus,nucleus accumbeus, and putamen. Trace levels were found in amygdala,caudate, cerebral cortex, frontal lobe, temporal lobe, hippocampus,spinal cord, uterus, stomach, pancreas, kidney, trachea, fetal heart,and fetal spleen.

When coexpressed with human Slo1, BK beta 4 causes inactivation (FIG.7). At a given calcium concentration, the rate of inactivation increaseswith increasing depolarization. Because this inactivation limits thetimecourse of potassium efflux through Slo channels, BK beta 4expression may exert a profound effect on how the slo currents are ableto modulate excitability and secretion.

1. An isolated nucleic acid encoding a beta subunit polypeptide of apotassium channel, wherein the beta subunit polypeptide: (i) forms, withfour alpha subunit polypeptides, a slo potassium channel; and (ii)comprises an amino acid sequence that has greater than 80% identity toSEQ ID NO: 5, and wherein said beta subunit polypeptide slows activationof the slo current.
 2. The isolated nucleic acid of claim 1, wherein thenucleic acid encodes a beta subunit polypeptide that specifically bindsto polyclonal antibodies generated against a polypeptide comprising anamino acid sequence of SEQ ID NO:
 5. 3. The isolated nucleic acid ofclaim 1, wherein the nucleic acid encodes a polypeptide comprising anamino acid sequence of SEQ ID NO:5.
 4. The isolated nucleic acidsequence of claim 1, wherein the nucleic acid comprises a nucleotidesequence of SEQ ID NO:6.
 5. An expression vector comprising the nucleicacid of claim
 1. 6. An isolated host cell transfected with the vector ofclaim 5.